PROJECT SUMMARY Background: Insulin resistance (IR), a hallmark of diabetes, is associated with elevated levels of circulating advanced glycation endproducts (AGEs) and their accumulation in adipose tissue. AGEs are the byproduct of non-enzymatic binding of sugars and/or lipids to proteins, a process that is prevented by specialized enzymes such as glyoxalase 1 (GLO1). Highly processed foods, characteristic of ?Western? diets, are a major source of exogenous AGEs and may be linked to the raise in incidence of obesity and diabetes. AGE signaling is primarily mediated by the receptor for advanced glycation endproducts (RAGE), which in turn interacts in the cytoplasm with diaphanous 1 (DIAPH1) to initiate downstream cellular signaling. Thus, determining whether AGE-induced IR requires the RAGE/DIAPH1 signaling axis may identify novel therapeutic targets to treat or prevent insulin resistance and diabetes. Hypothesis: We hypothesize that excessive AGEs, acting along the RAGE/DIAPH1 axis, induce insulin resistance via unknown mechanisms which I aim to unveil. Thus, inhibition of the RAGE/DIAPH1 is anticipated to prevent AGE-induced metabolic impairment. Furthermore, since GLO1 metabolizes AGE precursors, overexpression of GLO1 is hypothesized to enhance AGE-precursor detoxification leading to lower levels of circulating and tissue accumulated AGEs, and therefore protect against AGE-induced insulin resistance. Approach: Preliminary data suggest that carboxymethyllysine (CML-AGE), a prototypic AGE, impairs glucose metabolism without altering insulin secretion, likely by accumulating in metabolically active tissues and altering insulin sensitivity. What is not known is 1) whether CML-AGE differentially affects metabolically-relevant organs, 2) whether it directly affects proteins involved in the insulin signaling cascade or 3) if it requires the RAGE/DIAPH1 signaling axis to exert its detrimental metabolic effects. To test these hypotheses, we will treat lean, male and female wild type mice with CML-AGE either acutely or chronically and determine the mechanisms by which CML-AGE induces IR. Mice devoid of RAGE and/or DIAPH1 will be used to test whether CML-AGE- induced IR requires the RAGE/DIAPH1 signaling axis. We will also determine whether increasing AGE detoxification by overexpressing Glo1 in mice protects from obesity, IR and metabolic dysregulation. The translational relevance of our findings in mice to humans will be assessed by measuring AGEs, GLO1, RAGE, DIAPH1 and inflammatory markers in subcutaneous human adipose tissue samples derived from the non- diabetic patients before and after weight loss surgery which is associated with improved insulin sensitivity. Significance: Results from these studies will likely establish a causal link between AGEs and impaired metabolic homeostasis and have the potential to identify two independent approaches to inhibit/suppress AGE activity that if successful, may provide novel therapeutic targets for the treatment of insulin resistance and diabetes.